NF-kappaB includes a family of signal-activated transcription factors that normally regulate responses to injury and infection but which are aberrantly activated in many carcinomas. Cumulative evidence implicates NF-kappaB in cell survival, inflammation, angiogenesis, spread and therapeutic resistance during tumor development, progression and metastasis of carcinomas. Non-specific natural and synthetic agents that inhibit NF-kappaB have demonstrated activity and safety in prevention or therapy. NF-kappaB-activating kinases and the proteasome are under investigation for targeted prevention and therapy of carcinoma. We completed our phase I clinical trial of proteasome inhibitor bortezomib with reirradiation for patients with recurrent HNSCC (01-C-0104). Correlative studies revealed that treatment significantly enhanced apoptosis with inhibition of nuclear RELA, but other NF-kappaB subunits, ERK1/2, and STAT3 were variably or not affected, and tumor progression was often observed within 3 months. Overall, 5/17 patients treated at the initial dose level demonstrated partial responses. Accrual of 6 patients has been completed at the 0.9mg/m2 dose level, defining it as the maximally tolerated dose with a new schedule that provides a two week break from drug treatment. Studies in HNSCC cell lines, indicated that bortezomib partially inhibits basal activation of NF-kappaB1/RELA, but not NF-kappaB2/RELB, or MAPK-AP-1 activation. We conclude that although bortezomib inhibits activation of subunits of the canonical pathway, it does not block nuclear activation of the noncanonical NF-kappaB or MAPK-AP-1 or STAT3 prosurvival signal pathways, which may contribute to the heterogeneous responses observed in HNSCC. Combination of bortezomib with MAPK JNK inhibitor in vitro showed increased activity, indicating potential of combining bortezomib with other signal pathway inhibitors. An collaborative trial with NCI and University of Pittsburgh NCI SPORE investigators combining bortezomib to inhibit NF-kB, with Epidermal Growth Factor Receptor inhibitor antibody cetuximab to inhibit MAPK and STAT3, was initiated (NIH protocol 08-C-0071). 7 patients were accrued, with dose escalation of 3 patients each at 0.7,1.0, and one at 1.3mg/m2 without dose limiting toxicity. Preliminary assessment indicated a modest complete responses rate in 3/7 subjects, with shorter than expected disease free survival, prompting closure of accrual. ECOG 1304, a phase II study of bortezomib alone or in combination with irinotecan is under data analysis. Studies defining the role of upstream kinases TAK1, CK2, IKKalpha and beta and PKA as key signal activators of NF-kB RELA transactivation were completed and manuscripts were published or in preparation.